Chromane Derivatives from Underground Parts of Iris tenuifolia and Their In Vitro Antimicrobial, Cytotoxicity and Antiproliferative Evaluation

Phytochemical investigation of the ethanol extract of underground parts of Iris tenuifolia Pall. afforded five new compounds; an unusual macrolide termed moniristenulide (1), 5-methoxy-6,7-methylenedioxy-4-O-2′-cycloflavan (2), 5,7,2′,3′-tetrahydroxyflavanone (3), 5-hydroxy-6,7-dimethoxyisoflavone-2′-O-β-d-glucopyranoside (9), 5,2′,3′-dihydroxy-6,7-dimethoxyisoflavone (10), along with seven known compounds (4–8, 11–12). The structures of all purified compounds were established by analysis of 1D and 2D NMR spectroscopy and HR-ESI-MS. The antimicrobial activity of the compounds 1–3, 5, 9, and 10 was investigated using the agar diffusion method against fungi, Gram-positive and Gram-negative bacteria. In consequence, new compound 3 was found to possess the highest antibacterial activity against Enterococcus faecalis VRE and Mycobacterium vaccae. Cell proliferation and cytotoxicity tests were also applied on all isolated compounds and plant crude extract in vitro with the result of potent inhibitory effect against leukemia cells. In particular, the newly discovered isoflavone 10 was active against both of the leukemia cells K-562 and THP-1 while 4–6 of the flavanone type compounds were active against only THP-1.


Structure Elucidation
The ethanol extracts from the underground parts of I. tenuifolia were subjected to repeated column chromatography followed by crystallizations leading to the isolation of five unprecedented chromane derivatives.
Compound 1 was isolated as white crystal. HR-ESI-MS showed an ion peak at m/z 453.1409 [M + H] + corresponding to a molecular formula of C21H24O11. Its 1 H NMR spectrum acquired in DMSO-d6 (Table 1) showed resonances for meta-coupled aromatic protons at δH 6.14 (1H, J = 2.0 Hz) and δH 5.95 (1H, J = 2.0 Hz), two olefinic protons at δH 6.94 and δH 5.75, methylene signals between δH 3.21-1.81 and number of oxygenated protons between δH 5.50-3.08 corresponding to hydroxy groups as well as oxymethines. The presence of a 2,5,7-trisubstituted chromane-4-one was identified by analysis of HMBC correlations observed for the meta-coupled aromatic doublets H-6 and H-8 as well as methylene signals H-2 and H-3 ( Figure 2a). Moreover, the 1 H NMR spectrum exhibited a signal of one chelated hydroxyl group (δH 12.04), which is characteristic downfield shift of a hydroxyl group at C-5 and a carbonyl group at C-4. In addition, the presence of a hydroxyl group at C-5 was supported by HMBC correlations from 5-OH (δH 12.04) to C-5 (δC 163.1),

Structure Elucidation
The ethanol extracts from the underground parts of I. tenuifolia were subjected to repeated column chromatography followed by crystallizations leading to the isolation of five unprecedented chromane derivatives.
Compound 1 was isolated as white crystal. HR-ESI-MS showed an ion peak at m/z 453.1409 [M + H] + corresponding to a molecular formula of C 21 H 24 O 11 . Its 1 H NMR spectrum acquired in DMSO-d 6 ( Table 1) showed resonances for meta-coupled aromatic protons at δ H 6.14 (1H, J = 2.0 Hz) and δ H 5.95 (1H, J = 2.0 Hz), two olefinic protons at δ H 6.94 and δ H 5.75, methylene signals between δ H 3.21-1.81 and number of oxygenated protons between δ H 5.50-3.08 corresponding to hydroxy groups as well as oxymethines. The presence of a 2,5,7-trisubstituted chromane-4-one was identified by analysis of HMBC correlations observed for the meta-coupled aromatic doublets H-6 and H-8 as well as methylene signals H-2 and H-3 ( Figure 2a). Moreover, the 1 H NMR spectrum exhibited a signal of one chelated hydroxyl group (δ H 12.04), which is characteristic downfield shift of a hydroxyl group at C-5 and a carbonyl group at C-4. In addition, the presence of a hydroxyl group at C-5 was supported by HMBC correlations from 5-OH (δ H 12.04) to C-5 (δ C 163.1), C-6 (δ C 97.3) and C-10 (δ C 103.4). HSQC, HMBC, and COSY data clearly revealed the existence of a glucose residue. Further analysis of the spin-spin couplings (J = 7.7 Hz) allowed the identification of β-D-glucose. Connectivity of sugar moiety with chromane ring was deduced through HMBC correlation from the anomeric proton H-1" (δ H 5.09) to C-7 (δ C 164.1) of aglycon. A remaining spin system observed on the COSY spectrum comprised a series of protons H-2 -H-6 . HMBC correlations from olefinic protons H-2 and H-3 to a carbonyl carbon at δ C 164.8 (C-1 ) allowed for establishing 6-hydroxy-2-hexenoic acid residue. Trans configuration of the double bond has been identified by a large coupling constant of 15. 6 Hz. This substructure has been linked to the aglycon on position C-2 by HMBC correlations from the H-5 and H-6 to a carbon at δ C 76.9 (C-2) and in turn H-3 to a carbon at δ C 70.9 (C-6 ). Interestingly, a macrolide structure was established via ester linkage between sugar residue and carbonyl carbon of hexenoic acid by strong heteronuclear long-range correlation from H-6" to C-1 . ROESY correlations between diastereotopic H-3 eq (β) and H-2 and H-6 indicated that these protons are on the same side of the chromane ring. In turn, H-3 ax showed ROESY correlation to 6 -OH. From the above findings, relative configuration at C-2 and C-6 has been established as β-configured ( Figure 2b). The absolute configuration at C-2 was found to be S as it showed positive and negative Cotton effects at 305 and 287 nm, respectively in the circular dichroism (CD) spectrum ( Figure S9, Supplementary Materials) [11,12]. Thus, the structure of compound 1 was established as a new type of macrolide, named moniristenulide, as shown in Figure 1.  3.08 m 70.2 C-3", C-6" H-5", 4"-OH -5" 3.71 t (10.4, 1.2) 74.4 C-1", C-3", C-4", C-6" H-4", H-6" a , H-6" b H-1", H-3", H-6" a 6" a 4.36 brd (11.5) 63.2 C-1 , C-1", C-5" H-5", H-6" b H-2, H-5", H-6" b , 4"-OH 6" b 4.06 brd (10.9) C-1 , C-5" H-5", H-6" a -5-OH 12.04 s -C-4, C-5, C-6, C-10 --6 -OH 5.14 d (5.9) -C-2, C-5 , C-6 H-6 H-3 ax 2"-OH 5.50 d (5.1) -C-1", C-2", C-3" H-2" H-2" 3"-OH 5.25 d (5.1) -C-2", C-3", C-4" H-3" -4"-OH 5.36 d (5.1) -C-3", C-4", C-5" H-4" H-4", H-6" a H-3eq (β) and H-2 and H-6′ indicated that these protons are on the same side of the chromane ring. In turn, H-3ax showed ROESY correlation to 6′-OH. From the above findings, relative configuration at C-2 and C-6′ has been established as β-configured ( Figure 2b). The absolute configuration at C-2 was found to be S as it showed positive and negative Cotton effects at 305 and 287 nm, respectively in the circular dichroism (CD) spectrum ( Figure S9, Supplementary Materials) [11,12]. Thus, the structure of compound 1 was established as a new type of macrolide, named moniristenulide, as shown in Figure 1.   and one methoxy singlet at δ H 4.09 (3H, s). Analysis of 13 C NMR spectrum combined with HSQC allowed for identifying the existence of 17 carbon atoms, including 12 sp 2 carbons, two methylene carbons at δ C 26.5 and 100.8, two methine carbons at δ C 67.5 and 62.4, and one methoxy group at δ C 60.1 ( Table 2). The COSY correlations of four aromatic protons at δ H 7.33, 7.20, 6.89 and 6.87 (H-3 -H-6 ) along with the respective HMBC correlations confirmed a presence of disubstituted aromatic B-ring, while the remaining sharp singlet at δ H 6.08 (H-8) described a typical penta-substituted aromatic A-ring. Furthermore, the COSY experiment showed another spin system from H-2 to H-4 and indicated a CH-CH 2 -CH sequence. Additionally, the key HMBC correlation from H-6 (δ H 7.33) to C-2 (δ C 67.5) revealed the flavan (2-phenylchromane) skeleton ( Figure 3). The HMBC correlation from methine H-4 (δ H 5.64) to C-2 (δ C 153.6) and their chemical shift indicated their linkage through an oxygen bridge. All these data deduced that compound 2 has a 4-O-2 -cycloflavan as a partial structure. The remaining two doublet signals at δ H 5.81 and 5.76 had one bond correlation to a carbon atom at δ C 100.8, which were assigned as a methylenedioxy group. These methylenedioxy doublet was connected to a flavan core by key HMBC correlations to C-6 and C-7. The location of methoxy group (δ H 4.09) at C-5 was suggested by HMBC correlations. On the basis of the above evidence, the planar structure of compound 2 was elucidated as 5-methoxy-6,7-methylenedioxy-4-O-2 -cycloflavan and considered as an unprecedented natural product. This spectroscopic data of compound 2 is somewhat similar to the literature values for the known 4-O-2 -cycloflavan core structures, possessing a different substitution pattern in aromatic A-and B-rings [7,13,14].  (Table 2). Partial structure 2,5,7-trisubstituted chromane-4-one was deduced from the analysis of protons H-2 and H-3, which were existing in an AMX spin system ( Figure 3). In addition, hydroxyl groups at C-5 and C-7 were supported by HMBC correlations from 5-OH (δ H 12.12) to C-5 (δ C 163.5), C-6 (δ C 95.8) and C-10 (δ C 101.7) and from 7-OH to C-6 (δ C 95.8) and C-7 (δ C 166.6). In addition, a COSY correlation of three aromatic protons at δ H 6.69, 6.79, 6.88 along with HMBC correlations from proton H-4 (δ H 6.79) to C-2 (δ C 142.6), C-3 (δ C 145.2) and from proton H-6 (δ H 6.88) to C-2 (δ C 74.0), C-2 (δ C 142.6), and C-4 (δ C 115.2) revealed a presence of a 2,3-dihydroxyphenyl moiety (ring-B) and altogether confirmed the flavanone structure. This was further supported by key longrange heteronuclear correlations from the methine proton H-2 (δ H 5.70) to C-2 (δ C 142.6). The position of the remaining two hydroxyl protons at δ H 9.51 and 8.71 were assigned to C-3 and C-2 respectively, due to observed HMBC correlations. Spectral data of 3 possess close similarity to those for the known compounds 5 and 6 [15]. The only difference was observed for the substitution on C-7, where methoxy group in 6 and methylenedioxy group in 5, while it was replaced by hydroxy group in 3. This was supported by HMBC correlations from 7-OH to C-6, C-7, and C-8 in 3 (Figure 3). Table 2. 1 H NMR (500 MHz) and 13 C NMR (125 MHz) data of compounds 2, 3, 9, and 10 (δ in ppm, J in Hz). Position 2 * 3 ** 9 ** 10 *     Furthermore, the known compounds were readily identified by means of HR-ESI-MS, 1D, and 2D NMR data as well as in comparison with those previously reported in the literature: compound 4 as 5,2′-dihydroxy-6,7-methylenedioxyflavanone [15], 5 as 5,2′,3′- The absolute configuration at C-2 of compound 3 was determined as 2S based on its CD spectrum ( Figure S25, Supplementary Materials), which displayed a positive Cotton effect at 325 nm and a negative one at 283 nm [11,12]. Consequently, compound 3 was elucidated as (2S)-5,7,2 ,3 -tetrahydroxyflavanone, an undescribed member of a flavanone group of natural products.  Figure 3). The remaining aromatic signal appeared as a sharp singlet at 6.64 (1H, s) together with chelated hydroxyl group at δ H 12.68 suggested a typical penta-substituted aromatic A-ring. The singlet was assigned on C-8 according to HMBC correlation from H-8 to C-9 (δ C 149.2) and C-7 (δ C 158.3). The chelated hydroxyl group was positioned at C-5 by means of HMBC. In addition, a characteristic isoflavonoid signal for H-2 was appeared at δ H 8.44. The isoflavone nature was supported by long-range correlations from H-2 (δ H 8.44) to C-4 (δ C 180.5), C-9 (δ C 149.2), and C-1 (δ C 120.0) in the HMBC spectrum. Furthermore, two methoxyl singlet signals were apparent at δ H 3.79 and 3.94, and they were located at C-6 and C-7 due to HMBC correlations between 6-OCH 3 and C-6 (δ C 128.3), as well as 7-OCH 3 and C-7 (δ C 158.3). Moreover, a series of COSY cross signals comprising six protons in the range of δ H 3.0-4.0, four hydroxy protons δ H 4.57-5.05, as well as a doublet at δ H 4.89, revealed the presence of a glucose moiety (H-1" to H-6") [16]. The HMBC correlation from H-1" (δ H 4.89) to C-2 (δ C 155.1) revealed the sugar moiety was located at C-2 of aglycone. The coupling constant of anomeric proton J = 7.80 Hz indicated that the sugar was β-oriented. The 1 H and 13 C NMR spectroscopic data of aglycone of 9 were comparable with the literature values for the irilin A [17,18]. The only difference was occurred on C-2 , where OH group of irilin A was replaced by glucopyranosyl in 9. Finally, the structure of compound 9 was elucidated as 5-hydroxy-6,7dimethoxyisoflavone-2 -O-β-D-glucopyranoside, an unprecedented natural product.
Compound 10 was isolated as yellow crystal. HR-ESI-MS showed the [M + H] + peak at m/z 331.0825, corresponding to the molecular formula C 17 H 14 O 7 . 1 H NMR, 13 C NMR, and HSQC data of compound 10 closely resembled to that of 9, differing only on the absence of signals corresponding to a sugar moiety and different pattern on aromatic signals of B-ring ( Table 2). The COSY correlation of a triplet at δ H 6.92 (1H, t, J = 7.9 Hz) with two doublets of doublets at δ H 7.03 (1H, dd, J = 1.3, 7.9 Hz) and 6.71 (1H, dd, J = 1.5, 7.8 Hz), and their HMBC correlations clearly indicated ortho-dihydroxyl substitution on B-ring. The position of the hydroxyl groups, which were appeared as two singlets in the 1 H NMR spectrum at δ H 8.48 and 6.09, were assigned at C-2 and C-3 , due to the HMBC correlations ( Figure 3). Therefore, the structure of 10 was solved as 5,2 ,3 -trihydroxy-6,7-dimethoxyisoflavone, which is an undescribed natural product so far.

Antimicrobial Activity
The newly discovered compounds 1, 2, 3, 9, and 10 were tested together with the known compound 5, which is the major component of the roots of this plant [15] for their antifungal and antibacterial activities against three fungi and eight bacterial strains (Table 3) using the agar diffusion method. All tested compounds possessed weak to moderate activity against vancomycin resistant (VRE) Enterococcus faecalis. Compounds except 1 and 9 exhibited weak and moderate activities against Bacillus subtilis and Mycobacterium vaccae, respectively, compared to ciprofloxacin. Compounds 3, 5, and 10, which contain ortho-dihydroxyl groups in B-ring at positions 2 and 3 , were most active against bacterial strains. This supports the evidence that the ortho -dihydroxyl structural fragment in B-ring is important for antimicrobial activity [22]. Compound 2, a new cycloflavan, demonstrated activity against B. subtilis, E. faecalis, and M. vaccae. Interestingly, the new compounds 1 and 9, which have a glucose unit in their structure, showed selective activity only against E. faecalis VRE. As for the fungi, the compound 5 showed moderate activity against Penicillium notatum, and compounds 5 and 10 showed weak activity against Candida albicans. It is noteworthy that compound 5, which contains a methylenedioxy group, in addition to the ortho-dihydroxyl groups in B-ring, showed broad activity against eight microorganisms out of eleven. However, no inhibition was observed with the tested compounds against bacterial strains of Escherichia coli and Pseudomonas aeruginosa (SG137 B7) or against the fungus Sporobolomyces Salmonicolor. This is the first report on the antimicrobial activity of the tested compounds.

Antiproliferative and Cytotoxic Activities
Using HUVEC, K-562, THP-1, A549, and HeLa cell lines, the antiproliferative activities and the cytotoxicity of the isolated compounds (1-12) along with plant raw extract were evaluated in vitro ( Table 4). The plant extract possessed antiproliferative effects against leukemia cell lines and a cytotoxic effect on Hela cells. With the exception of 12, the compounds tested in this study were chromane derivatives. Compounds 4-6 and 10 showed inhibition effects against all of the applied cancer cell lines and exhibited potential antiproliferative activities against the THP-1 cell line with GI 50 values at 16.0, 16.5, 16.9, and 9.1 µM, respectively. Moreover, compound 10 demonstrated the most potent activity (GI 50 = 7.6 µM) against the K-562, followed by compounds 2 and 3 with GI 50 value at 31.5 and 32.3 µM. Compound 3, the tetrahydroxy flavanone, showed selective antiproliferative inhibition effect against the K-562 cells only. These results are in a good agreement with previous studies and supports the evidence that the ortho-dihydroxyl structural fragment in B-ring is very important for anticancer activity [7,[23][24][25]. Furthermore, compound 2, the cycloflavan, exhibited significant inhibitory activities against cell lines HUVEC and THP-1 with a GI 50 values at 35.2 and 29.2 µM and showed the cytotoxic effect on HeLa cells with CC 50 value at 42.6 µM among the tested compounds. Besides that, compound 10 showed a similar moderate inhibition effect (GI 50 = 35.8 µM) on HUVEC cells. All the flavonoids examined showed their low toxicity with CC 50 values of more than 100 µM on HeLa cells. In cases of compounds 7 and 8 which do not have ortho-dihydroxy substitutions on the A and B rings, reduced or no inhibitory effects were found compared to other compounds mentioned above. Compound 11 showed slightly increased activity against K-562 and moreover against THP-1 and A549 cell lines compared to compound 7. Thus, it supports the relevance of the C-2 and C-3 double bond in a flavonoid structure [23]. In contrast to 10, compound 9, which differs in its structure in the presence of a sugar moiety and a dehydroxylation on the B-ring, became completely not effective. Interestingly, compound 1, which also has a sugar residue in its structure, did not show any activity up to 100 µM against all human cancer cell lines either. Hence, the results are consistent with previous research that flavonoids glycosides were generally not effective against multiple cancer cell lines [23]. Compound 12 showed neither an antiproliferative effect nor a cytotoxicity within the tested range against all applied cell lines. The GI 50 and CC 50 values with 95% confidence intervals (CI 95%): 1-10 (very strong); 11-20 (strong); 21-50 (moderate); 51-100 (weak), and >100 (ineffective); n.d-not determined.

General Experimental Procedures
Solvents and reagents were purchased from Sigma-Aldrich, Deisenhofen, Germany and Qingdao Marine Chemical, China. The optical data were measured using a digital JASCO P-2000 polarimeter (Jasco, Pfungstadt, Germany). The CD spectrum was recorded on a JASCO J810 spectropolarimeter (Jasco, Pfungstadt, Germany). Ultraviolet-visible (UV-Vis) data were extracted from diode array detector (DAD) data obtained during high performance liquid chromatography-electrospray ionization-high resolution mass spectrometry (HPLC-ESI-HRMS) experiments.
NMR spectra were recorded at 298 K on 500 MHz Bruker Avance III HD spectrometer (Bruker Biospin, Rheinstetten, Germany), equipped with cryoplatforms and TCI cryoprobes (5 mm). Spectrometer control and data processing were accomplished using Bruker Topspin ver.3.2 (Bruker Biospin, Rheinstetten, Germany), and standard pulse programs were used. NMR signals were referenced to the respective solvent signals at δ H 2.50 and δ C 39.53 for hexadeuterodimethyl sulfoxide DMSO-d 6 and δ H 7.26 and δ C 77.06 for deuterochloroform CDCl 3 . HPLC-ESI-HRMS spectra were recorded on an Agilent Infinity 1260, consisting of a combined degasser and quaternary pump, column oven, autosampler, and DAD. The DAD was coupled to a Bruker Compact quadrupole time-of-flight (QTOF) mass spectrometer (Bruker Daltonics, Bremen, Germany). Both devices were controlled by Bruker Compass ver.1.9 (Bruker Daltonics, Bremen, Germany). For HPLC separation, an Agilent Zorbax C-18 SB column (3.5 µm, 4.6 × 150 mm i.d.) was used. The mass spectrometer was operated, depending on the analyte, either in positive or negative ionization mode, employing an electrospray ionization (ESI) source. The standard settings for small molecule analysis, as provided with Bruker Compass, were used. Column chromatographic separations were performed on silica gel (200-300 mesh, Merck, Darmstadt, Germany and Qingdao Marine Chemical, China) and Sephadex LH-20 (Pharmacia Fine Chemical, Uppsala, Sweden). Thin-layer chromatography (TLC) was performed on pre-coated TLC plates with silica gel 60 F 254 (Merck, Darmstadt, Germany). Spots were detected under UV absorption (λ max 254 and 364 nm) by spraying with 1% methanolic diphenylboric acid-β-ethylaminoester, 5% ethanolic polyethylene glycol or under visible light by spraying with 5% ethanolic sulfuric acid and 1% acidified methanolic vanillin.

Plant Material
Rhizomes and roots of I. tenuifolia were collected in September 2016 from Khurmen Sum of South Gobi province of Mongolia. It was identified by Urgamal Magsar, a botanist of the Institute of General and Experimental Biology, Mongolian Academy of Sciences, where voucher specimens (It 0916) of the plant have been deposited.

Antiproliferation and Cytotoxicity Assays
Compounds (1-12) were assayed against human umbilical vein endothelial cells (HU-VEC), human chronic myeloid leukemia cells (K-562), human acute monocytic leukemia cells (THP-1), and human lung carcinoma cells (A549) for their antiproliferative effects and against human cervix carcinoma cells (HeLa) for their cytotoxic effect. The antiproliperative and cytotoxic effects were tested via CellTiter-Blue and methylene blue assay as previously described [27]. In this assay, K-562 (DSM ACC 10), THP-1 (DSM ACC 16), and HeLa (DSM ACC 57) were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (Cambrex 12-167F) while HUVEC (ATCC CRL-1730) and A549 (DSM ACC 107) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Cambrex 12-614F). Cells that were grown in the appropriate cell culture medium were supplemented with 10 mL/L ultraglutamine 1 (Cambrex 17-605E/U1), 550 µL/L (50 mg/mL) gentamicin sulfate (Cambrex 17-518Z), and 10% heat inactivated fetal bovine serum (GIBCO Life Technologies 10270-106) at 37 • C. The tested compounds were dissolved in DMSO, and the cells were seeded in 96-well plates at a density of 1 × 10 4 cells/well. As for the antiproliferative effect of the compounds, the cells were incubated for 72 h, and GI 50 values were evaluated to be defined as the concentration causing 50% inhibition of proliferation compared to the untreated control. With regard to the cytotoxic assay, HeLa cells were pre-incubated for 48 h without the test compounds. Then, the cells were exposed with different concentrations of compounds and incubated for 72 h. After that, the adherent HeLa cells were fixed by glutaraldehyde and stained with a 0.05% solutions of methylene blue (SERVA 29198) for 15 min. CC 50 was evaluated to be defined as the concentration required for the death of 50% of the cell monolayer as compared to control groups. Under our experimental conditions, the optical density measured from the CellTiter-Blue reagent and methylene blue assay is proportional to the number of viable cells. In this experiment, absorbances were measured at 570 nm against the reference wavelength of 600 nm (CellTiter-Blue assay) and at 660 nm (methylene blue assay). Doxorubicin (Adriamycin ® ) and imatinib (Gleevec ® ) were used as positive controls for HUVEC, K-562, and HeLa cells. A repeat determination has been conducted in all experiments, and four replicates were assayed. The calculations of the different values of GI 50 and CC 50 were performed with software Magellan version 3.00 (Tecan Trading AG, Maennedorf, Switzerland).
Notably, eight out of nine isolated flavonoids have a rare 2 ,3 -disubstituted configuration on the B-ring, out of which the compounds bearing ortho-dihydroxyl groups in B-ring, namely 3, 5, and 10, showed the broadest antimicrobial activity. On top of that, the molecules with methoxy or methylenedioxy substitution on the A-ring together with ortho-hydroxyl groups on the B-ring showed promising antiproliferative activities against leukemia cell lines in combination with low cytotoxicity, as shown for compounds 4-6 and 10.